This invention relates to peptide hormone receptors.
Peptide hormone receptors are important targets for drug research because a considerable number of diseases and other adverse effects result from abnormal receptor activity. High affinity, high specificity, non-peptide antagonists for peptide hormone receptors have been developed. These antagonists are therapeutically useful for decreasing receptor activation by endogenous hormones. Developing non-peptide agonists proved to be far more difficult.
One peptide hormone of interest, cholecystokinin (CCK), is a neuropeptide with two distinct receptors: CCK-A and CCK-B/gastrin (Vanderhaeghen et al., Nature, 257:604-605, 1975; Dockray, Nature, 264:568-570, 1976; Rehfeld, J. Biol. Chem., 253:4022-4030, 1978; Hill et al., Brain Res., 526:276-283, 1990; Hill et al., J. Neurosci., 10:1070-1081, 1990; Woodruff et al., Neuropeptides, (Suppl.) 19:57-64, 1991). The peripheral type receptor CCK-A is located in discrete brain nuclei and, in certain species, the spinal cord, and is also involved in gallbladder contraction and pancreatic enzyme secretion. The CCK-B/gastrin receptor is most abundant in the cerebral cortex, cerebellum, basal ganglia, and amygdala of the brain, as well as in parietal cells of the gastrointestinal tract. CCK-B receptor antagonists have been postulated to modulate anxiety, panic attacks, analgesia, and satiety (Ravard et al., Trends Pharmacol. Sci., 11:271-273, 1990; Singh et al., Proc. Natl. Acad. Sci. U.S.A., 88:1130-1133, 1991; Faris et al., Science, 219:310-312, 1983; Dourish et al., Eur.J.Pharmacol., 176:35-44, 1990; Wiertelak et al., Science, 256:830-833, 1992; Dourish et al., Science, 245:1509-1511, 1989).
Applicants have developed a systematic screening assay for identifying non-peptide agonists specific to peptide hormone receptors. The assay is based on applicants"" recognition that a peptide hormone receptor having the capability of amplifying the intrinsic activity of a ligand is useful as a screening vehicle to identify receptor-specific agonists. In addition, a receptor with a signaling activity higher than the corresponding human wild-type basal level of signaling activity is especially useful for detecting a reduction in activity induced by an inverse agonist. In both cases, the receptor amplifies the signal generated when the ligand interacts with its receptor, relative to the signal generated when the ligand interacts with a human wild-type receptor. Thus, forms of a receptor with the ability to amplify receptor signaling are useful for efficiently screening positive and inverse non-peptide agonists to the corresponding human wild-type form of the receptor.
Accordingly, the invention features a method for determining whether a candidate compound is a non-peptide agonist of a peptide hormone receptor. In this method, a candidate compound is exposed to a form of the peptide hormone receptor which has a greater, or an enhanced, ability to amplify the intrinsic activity of a non-peptide agonist (hereafter an xe2x80x98enhanced receptorxe2x80x99). The second messenger signaling activity of the enhanced receptor is measured in the presence of the candidate compound, and compared to the second messenger signaling activity of the enhanced receptor measured in the absence of the candidate compound. A change in second messenger signaling activity indicates that the candidate compound is an agonist. For example, an increase in second messenger signaling activity indicates that the compound is either a full or partial positive agonist; a decrease in second messenger signaling activity indicates that the compound is an inverse (also termed a xe2x80x98negativexe2x80x99) agonist.
By xe2x80x9cintrinsic activityxe2x80x9d is meant the ability of a ligand to activate a receptor, i.e., to act as an agonist. By xe2x80x98amplifyxe2x80x99 is meant that the signal generated when the ligand interacts with the enhanced receptor is either higher for a positive agonist, or lower for an inverse agonist, than the signal produced when the same ligand interacts with a corresponding non-enhanced receptor, e.g., a wild-type human receptor. A xe2x80x98non-enhanced receptorxe2x80x99, for the purposes of this invention, is a wild-type human receptor for the peptide hormone of interest. By xe2x80x9ccorrespondingxe2x80x9d is meant the same type of peptide hormone receptor albeit in another form, e.g., a constitutively active mutant receptor. By way of example, the corresponding wild-type form of a constitutively active mutant CCK-B/gastrin receptor would be a wild-type CCK-B/gastrin receptor; the human CCK-B/gastrin receptor is the corresponding human form of the rat CCK-B/gastrin receptor.
Examples of enhanced receptors include synthetic mutant receptors, e.g., constitutively active mutant receptors; other mutant receptors with normal basal activity which amplify the intrinsic activity of a compound; naturally-occurring mutant receptors, e.g., those which cause a disease phenotype by virtue of their enhanced receptor activity, e.g., a naturally-occurring constitutively active receptor; and either constitutively active or wild-type non-human receptors, e.g., rat, mouse, mastomys, Xenopus, or canine receptors or hybrid variants thereof, which amplify an agonist signal to a greater extent than does the corresponding wild-type human receptor. An enhanced receptor may, but does not always, have a higher basal activity than the basal activity of a corresponding human wild-type receptor. Methods for measuring the activity of an enhanced receptor relative to the activity of a corresponding wild-type receptor are described and demonstrated below.
Examples of peptide hormone receptors within the scope of the invention include, but are not limited to, receptors specific for the following peptide hormones: amylin, angiotensin, bombesin, bradykinin, C5a anaphylatoxin, calcitonin, calcitonin-gene related peptide (CGRP), chemokines, cholecystokinin (CCK), endothelin, follicle stimulating hormone (FSH), formyl-methionyl peptides, galanin, gastrin, gastrin releasing peptide, glucagon, glucagon-like peptide 1, glycoprotein hormones, gonadotrophin-releasing hormone, leptin, luteinizing hormone (LH), melanocortins, neuropeptide Y, neurotensin, opioid, oxytocin, parathyroid hormone, secretin, somatostatin, tachykinins, thrombin, thyrotrophin, thyrotrophin releasing hormone, vasoactive intestinal polypeptide (VIP), and vasopressin. An enhanced receptor can further embrace a single transmembrane domain peptide hormone receptor, e.g., an insulin receptor.
An xe2x80x9cagonistxe2x80x9d, as used herein, includes a positive agonist, e.g., a full or a partial positive agonist, or a negative agonist, i.e., an inverse agonist. An agonist is a chemical substance that combines with a receptor so as to initiate an activity of the receptor; for peptide hormone receptors, the agonist preferably alters a second messenger signaling activity. A positive agonist is a compound that enhances or increases the activity or second messenger signaling of a receptor. A xe2x80x9cfull agonistxe2x80x9d refers to an agonist capable of activating the receptor to the maximum level of activity, e.g., a level of activity which is induced by a natural, i.e., an endogenous, peptide hormone. A xe2x80x9cpartial agonistxe2x80x9d refers to a positive agonist with reduced intrinsic activity relative to a full agonist. As used herein, a xe2x80x9cpeptoidxe2x80x9d is a peptide-derived partial agonist. An xe2x80x9cinverse agonistxe2x80x9d, as used herein, has a negative intrinsic activity, and reduces the receptor""s signaling activity relative to the signaling activity measured in the absence of the inverse agonist. A diagram explaining the difference between full and partial agonists, inverse agonists, and antagonists is shown in FIG. 1 (see also Milligan et al., TIPS, 16:10-13, 1995).
Examples of peptide hormone receptor specific peptide agonists and non-peptide antagonists useful in the screening assay of the invention are described below. Non-peptide ligands include, but are not limited to, the benzodiazepines, e.g., azabicyclo[3.2.2]nonane benzodiazepine (L-740,093; Castro Pineiro et al., WO 94/03437). L-740,093 S and L-740,093 R refer to the S-enantiomer and the R-enantiomer of L-740,093, respectively. Where the peptide hormone receptor is a CCK-A or CCK-B/gastrin receptor, useful peptide agonists include, but are not limited to, gastrin (e.g., sulphated (xe2x80x9cgastrin IIxe2x80x9d) or unsulphated (xe2x80x9cgastrin Ixe2x80x9d) forms of gastrin-17, or sulphated or unsulphated forms of gastrin-34), or cholecystokinin (CCK) (e.g., sulfated CCK-8 (CCK-8 s), unsulphated CCK-8 (CCK-8d), CCK-4, or pentagastrin (CCK-5)). Full agonists of the CCK-B/gastrin receptor include, but are not limited to, CCK-8s, and more preferably gastrin (gastrin I).
In contrast, an xe2x80x9cantagonistxe2x80x9d, as used herein, refers to a chemical substance that inhibits the ability of an agonist to increase or decrease receptor activity. A xe2x80x98fullxe2x80x99, or xe2x80x98perfectxe2x80x99 antagonist has no intrinsic activity, and no effect on the receptor""s basal activity (FIG. 1). Peptide-derived antagonists are, for the purposes herein, considered to be non-peptide ligands.
The invention also features a method of isolating a form of a peptide hormone receptor suitable for detecting agonist activity of a non-peptide ligand. The method involves (a) exchanging a region of a functional domain of a first peptide hormone receptor with a corresponding region of a functional domain of a second peptide hormone receptor, the functional domain being selected from the group consisting of an intracellular loop and adjacent parts of a transmembrane domain; and (b) measuring the ability of the first peptide hormone receptor to amplify an agonist signal relative to a corresponding wild-type human receptor, a greater amplification in the first peptide hormone receptor would indicate that the first peptide hormone receptor is suitable for detecting agonist activity in a non-peptide ligand. The corresponding region can be between one and ten amino acids, e.g., a block of five to ten amino acids, or up to thirty or a hundred amino acids in length. The first and second peptide hormone receptors are preferably linked to different second messenger pathways. Those skilled in the art know which particular amino acids of the peptide hormone receptors are considered to be within extracellular, intracellular (cytoplasmic), or transmembrane regions of the receptor. For example, extracellular, intracellular, and transmembrane regions of the CCK-B/gastrin receptor are determined by sequence alignment with other receptors (FIG. 2), or by hydropathy analysis (Baldwin, EMBO J., 12:1693-1703, 1993). Conformation receptor modelling is described further below.
Another method of isolating a form of a peptide hormone receptor suitable for detecting agonist activity in a non-peptide ligand involves (a) constructing a series of mutant forms of the receptor by replacing an original amino acid with another amino acid, i.e., a replacement amino acid; and (b) measuring the ability of the first peptide hormone receptor to amplify an agonist signal relative to the corresponding wild-type human receptor. An amplification in the first peptide hormone receptor would indicate that the first peptide hormone receptor is suitable for detecting agonist activity in a non-peptide ligand. The replaced amino acid can lie in an intracellular domain of the receptor or in a region of a transmembrane domain flanking an intracellular portion of the receptor, e.g., the intracellular domain-proximal half of the transmembrane domain, or within, e.g., 8 or 10 amino acids of the intracellular domain. The replacement amino acid can be of the same type in each of the mutant constructs, or various types of amino acids can be substituted at random. The replacement amino acid can be of the same or a different charge from the original amino acid, e.g., a negative amino acid can be exchanged for a positive amino acid, a positive amino acid can be exchanged for a negative amino acid, or a positive or negative amino acid can be exchanged for a neutral amino acid. Preferably, the replacement amino acid is glutamine, glutamic acid, aspartic acid, or serine.
Other terms used in the various embodiments of the invention will be understood from the following definitions. For example, by a xe2x80x9cpeptide hormonexe2x80x9d is meant a polypeptide that interacts with a target cell by contacting an extracellular receptor, i.e., a xe2x80x9cpeptide hormone receptorxe2x80x9d. A xe2x80x9cpeptidexe2x80x9d is used loosely herein to refer to a molecule comprised of amino acid residues that are connected to each other by peptide bonds. A xe2x80x9cmutant receptorxe2x80x9d is understood to be a form of the receptor in which one or more amino acid residues in the predominant receptor occurring in nature, e.g., in a naturally-occurring wild-type receptor, have been either deleted or replaced with a different type of amino acid residue. By a xe2x80x9cconstitutively active receptorxe2x80x9d is meant a receptor with a higher basal activity level than the corresponding wild-type receptor, where activity means the spontaneous ability of a receptor to signal in the absence of further activation by a positive agonist. The basal activity of a constitutively active receptor can also be decreased by an inverse agonist. A xe2x80x9cnaturally-occurringxe2x80x9d receptor refers to a form or sequence of the receptor as it exists in an animal, or to a form of the receptor that is synonymous with the sequence known to those skilled in the art as the xe2x80x9cwild-typexe2x80x9d sequence. Those skilled in the art will understand a xe2x80x9cwild-typexe2x80x9d receptor to refer to the conventionally accepted xe2x80x9cwild-typexe2x80x9d amino acid consensus sequence of the receptor, or to a xe2x80x9cnaturally-occurringxe2x80x9d receptor with normal physiological patterns of ligand binding and signaling. A xe2x80x9csecond messenger signaling activityxe2x80x9d refers to production of an intracellular stimulus (including, but not limited to, cAMP, cGMP, ppGpp, inositol phosphate, or calcium ion) in response to activation of the receptor, or to activation of a protein in response to receptor activation, including but not limited to a kinase, a phosphatase, or to activation or inhibition of a membrane channel.
xe2x80x9cSequence identity,xe2x80x9d as used herein, refers to the subunit sequence similarity between two nucleic acid or polypeptide molecules. When a given position in both of the two molecules is occupied by the same nucleotide or amino acid residue, e.g., if a given position (as determined by conventionally known methods of sequence alignment) in each of two polypeptides is occupied by serine, then they are identical at that position. The identity between two sequences is a direct function of the number of matching or identical positions, e.g., if 90% of the positions in two polypeptide sequences are identical, e.g., 9 of 10, are matched, the two sequences share 90% sequence identity. Methods of sequence analysis and alignment for the purpose of comparing the sequence identity of two comparison sequences are well known by those skilled in the art. xe2x80x9cBiological activityxe2x80x9d, as used herein, refers to the ability of a peptide hormone receptor to bind to a ligand, e.g., an agonist or an antagonist and to induce signaling.
The invention provides an efficient and rapid assay for identifying non-peptide agonists that interact with a peptide hormone receptor. The newly identified agonists can serve as therapeutics, or as lead compounds for further pharmaceutical research. Systematic chemical modifications can be made; their effects can be functionally assessed in enhanced receptors according to the method of the invention. By following such a development strategy the intrinsic activity of new agonists is optimized so as to provide useful therapeutics against diseases involving a peptide-hormone receptor.
Also embraced are the various mutant peptide hormone receptors disclosed herein, and their respective nucleic acid coding sequences. Plasmid manipulation, storage, and cell transformation are performed by methods known to those of ordinary skill in the art. See, e.g., Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY. 1988, 1995.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.
We first briefly describe the drawings.